One characteristic that contributes to Enterococcus faecalis's success as the second or third leading cause of hospital-acquired infection in the U.S. is its flexible metabolism, which enables survival under a wide variety of conditions. However, little is known about how E. faecalis regulates gene expression involved in employing alternative energy sources. The objective of this application is to elucidate the roles of an atypical B12 riboswitch and a response regulator involved in regulating ethanolamine utilization, a potentially valuable source of carbon and nitrogen in the host. Our central hypothesis is that a riboswitch located in an untranslated leader RNA in the ethanolamine-utilization operon, binds adenosylcobalamin (AdoB12), facilitating binding of a response regulator that leads to disruption of a terminator, and results in transcription of the downstream genes. The rationale for this research is that it elucidates a novel type of B12 riboswitch unlike any previously described in terms of structure, regulatory outcome (positive vs. negative), and most importantly, the additional involvement of a regulatory protein. The partner protein feature of this riboswitch implicates an expanded mechanistic diversity for these elements well beyond the current thinking. Aim #1 will identify the mechanism by which AdoB12 positively regulates the expression of the ethanolamine-utilization genes. The working hypothesis is that AdoB12 binds to the leader RNA inducing a change in the secondary structure that facilitates binding of the response regulator, EutRR. After establishing the transcriptional start site of the leader RNA, we will use in-line probing and SHAPE assays to demonstrate that AdoB12 binding causes structural change in the RNA, which increases its affinity for EutRR. Aim #2 will elucidate the mechanism by which EutRR regulates the expression of the eut genes. We postulate that the response regulator positively regulates gene expression by disrupting a terminator located just proximal to the start of the coding region. We will establish that EutRR is a positive regulator by showing loss of gene expression in a deletion mutant. Then we will show that EutRR has anti-terminator activity by a single-round in vitro transcription assay. As a result of the proposed investigations, the mechanism of an atypical B12 riboswitch will be uncovered. The research proposed is significant because it will contribute to efforts focused on exploiting riboswitches as possible antimicrobial targets in pathogenic bacteria. PUBLIC HEALTH RELEVANCE: The research proposed in this application will lead to greater understanding of how regulatory RNA's in a human bacterial pathogen control gene expression. Such knowledge is relevant to public health because it will contribute to efforts focused on exploiting these regulatory mechanisms as potential antimicrobial targets.